Bioassays are based on the interaction of at least one labelled biomolecule with an analyte (target) to be detected. The label acts as a marker indicating that a reaction has taken place between the target and a purposely chosen receptor or affinity molecule on the biomolecule that exclusively interacts with and binds to the target. The label can be measured using different techniques:                (i) optically by the measurement of the absorption of a dye or the fluorescent light emitted by fluorophores, or the luminescent light emitted by luminescent or chemiluminescent compounds, or measurement of turbidity caused by the light scattering of agglutinated latex particles;        (ii) radioactively by the measurement of radio isotopes;        (iii) electrochemically by the measurement of mediators or electroactive substances;        (iv) magnetically by the measurement of magnetic force; or.        (v) piezoelectrically by the measurement of changes in mass.        
Well known labels include enzymes as used in enzyme-linked immunosorbent assays (ELISAs) and radio isotopes used in radioimmunoassay; other label types include fluorophores, luminophores, chromophores, liposomes, latex particles in immuno agglutination assays, dyes, mediators, and gold particles.
The present invention is particularly, though not exclusively, concerned with labels that are detectable using optical techniques and with controlling the onset of maximum signal output so that readings can be taken and, if desired, repeated during a time period before the signal output decays or before the signal becomes dissipated through dilution and/or diffusion.
The purpose of the capsules in the present invention is two-fold: firstly, they serve as a vehicle or substrate for affinity molecules or receptors that are attached on the capsule surface and that are selected for their ability to specifically recognise and bind to a target molecule in a sample. Secondly, the capsules contain many molecules (107 to 109 or higher) of the signal precursor. Consequently, upon binding between a target molecule with one of the affinity molecules on a capsule surface and upon subsequent conversion of the signal precursor within the capsule from its latent form to its signal generating form, the presence of a single target molecule can be indicated by many tens of millions or even thousands of millions of detectable signal generating molecules. This is a very powerful amplification technique that can extend the limits of detection of a bioassay.
Capsules containing an organic signal-generating substance for use in bioassays are known from published international patent application no. WO02/12888 A2, the disclosure of which is incorporated herein by reference in its entirety. These known capsules are prepared by treating an uncharged solid organic signal-generating substance that has low water solubility or that is water insoluble (such as fluorescein diacetate (FDA)) with an aqueous solution of an amphiphilic substance (e.g., ionic detergent), The amphiphilic substance arranges itself on the surface of the solid signal-generating substance, imparting an electrical charge to its surface and rendering it susceptible to subsequent coating with a layer of a charged polyelectrolyte, followed by multiple alternating layers of oppositely charged polyelectrolytes. Polymer layers self-assemble onto the solid signal-generating substance (with its induced charge from the amphiphilic substance) by means of electrostatic layer-by-layer deposition, thus forming a multilayered polymeric shell around the solid core.
The coating step can also be carried out using a single layer of a substance bearing functional groups for covalent coupling of the coating layer, not using electrostatic deposition.
The thus-obtained capsules are modified for use in a bioassay by having affinity molecules attached to their surface, the affinity molecules being selected according to the type of target molecule that is to be detected.
In a first step of a detection method using the above-described capsules, a solution of target molecules is incubated with capsules modified with affinity molecules that specifically recognise the target molecules. The incubation is carried out over a time period sufficient to result in a target-affinity molecule complex; the affinity molecule remains bound to the capsule.
In a second step of the detection method, the resulting target-affinity molecule-capsule complexes are separated from capsules whose affinity molecules have not formed complexes with target molecules.
In a third step of the detection method, the capsules are disintegrated to release the signal-generating organic substance into solution, for example by treating them with an organic solvent such as an alcohol, a ketone, an ester, an ether, etc.
In the final step of the detection method, the signal that is generated by the released and dissolved signal-generating organic substance is detected and measured. The detected signal is related to the amount of the target molecules.
However, one of the disadvantages of this known detection method is that the disintegration of the capsules and the generation of the signal by the signal-generating organic substance is generally slow and can take varying amounts of time. This is undesirable because, in a lateral flow test using a liquid permeable membrane or a test using microfluidic channels, for example, the signal generating molecules are released into a solvent stream that is undergoing lateral flow. Hence, slow release of the signal generating molecules can lead to a detectable signal that is diffuse because it is spread by the flow of solvent.